Plant Cell, Tissue and Organ Culture 64: 145–157, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

145

Oxidative stress and physiological, epigenetic and genetic variability in

plant tissue culture: implications for micropropagators and genetic
engineers

Alan C. Cassells & Rosario F. Curry

Department of Plant Science, National University of Ireland, Cork, Ireland (∗ requests for offprints; Fax: +353-21-
274420; E-mail: [email protected])

Received 18 April 2000; accepted in revised form 1 December 2000

Key words: DNA repair, free radicals, genetic engineering, hyperhydricity, in vitro culture, juvenility, micro-
propagation, mutation, reactive oxygen species, somaclonal variation, tissue culture

Abstract
A number of well defined problems in physiological, epigenetic and genetic quality are associated with the culture
of plant cell, tissue and organs in vitro, namely, absence or loss of organogenic potential (recalcitrance), hyperhy-
dricity (‘vitrification’) and somaclonal variation. These broad terms are used to describe complex phenomena that
are known to be genotype and environment dependent. These phenomena affect the practical application of plant
tissue culture in plant propagation and in plant genetic manipulation. Here it is hypothesised much of the variability
expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant
tissues during explant preparation, and in culture, due to media and environmental factors. The characteristics of
these phenomena are described and causes discussed in terms of the known effects of oxidative stress on eukaryote
genomes. Parameters to characterise the phenomena are described and methods to remediate the causes proposed.

Abbreviations: AFLP – amplified fragment length polymorphism; FISH – fluorescent in situ hybridization;
HSP – heat shock proteins; PR-proteins – pathogenesis-related proteins; RFLP – restriction fragment length
polymorphism; ROS – reactive oxygen species

Introduction ‘nodal culture’ is preferred over proliferation of pre-

cocious axillary buds in shoot tip explants. The basis
Those using tissue culture for multiplication or trans- for this is that apical explants may give rise to basal
formation are concerned to produce microplants that explant callus from which adventitious buds may arise.
are ‘fit for the purpose’, that is, free of specified The latter has been associated in strawberry with ge-
diseases, vigorous, developmentally normal and ge- netic variability in the progeny (Jemmali et al., 1997).
netically true-to-type (Cassells, 2000a, b; Cassells et It is important to mention here that epigenetic and ge-
al., 2000). The exceptions are that the market may netic instability in the tissues used for Agrobacterium
exploit altered developmental characteristics, e.g. ju- transformation (somaclonal variation: see Jain et al.,
venility in herbaceous or woody plants where this 1998), that is expressed in adventitious shoots, may
results in greater productivity of the microplants when result in chimeral transformants (Cassells et al., 1987),
used for cutting production (George, 1993, 1996) or and in somaclonal variation in the background of the
where tissue protocols give earlier flowering (Cassells, transgenic lines (Sala et al., 2000) which may contrib-
2000a). In general, genotype-dependent, multiplica- ute to the silencing of trangenes (Matzke and Matzke,
tion via buds tends to be the preferred strategy to 1998).
maintain genetic stability (Figure 1; George, 1993). In human health the importance of oxidative stress
Proliferation of side shoots from axillary buds, termed has been long recognised in cancer and ageing studies
146

Figure 1. The methods of micropropagation least likely to produce plants with genetic variation (reproduced with permission; from George,
1993).

(Harman, 1956). It is also recognised how complex primary explants. Many factors associated with ab-
the underlying mechanisms and processes are (Hal- errations in plant tissue culture such as habituation,
liwell and Aruoma, 1993). The cellular mechanisms hyperhydricity (Gaspar, 1998) are caused by oxidat-
to manage stress, namely, constitutive and induced ive stresses (Keevers et al., 1995), such as high salt
production of radical scavengers, free radical and (McKersie and Leshem, 1994), water stress (Navari-
oxidised-protein enzymatic degradation pathways and Izzo et al., 1996), mineral deficiency (Elstner, 1991),
DNA repair mechanisms are highly conserved in all excess metal ions (Caro and Puntarulo, 1996) and
eukaryotes (Halliwell and Aruoma, 1993; McKersie possible over exposure to auxin (Droog, 1997).
and Leshem, 1994). Environmental and pathogen- Oxidative stress (Gille and Sigler, 1995; Bartosz,
induced stress have been investigated in detail in plants 1997) is defined as an imbalance in the pro- versus
in vivo (Bolwell et al., 1995; Baker and Orlandi, 1995; anti-oxidant ratio in cells and results in elevated levels
Doke et al., 1996; McKersie and Leshem, 1994). of pro-oxidants (ROS: reactive oxygen species; in-
Stress-like phenomena expressed in vitro and in mi- cluding superoxide, hydrogen peroxide hydroxyl, per-
croplants have been extensively described but less is oxyl and alkoxyl radicals) (Wiseman and Halliwell,
known about the underlying causal mechanisms (Ziv, 1996) which can cause cell damage (Sies, 1991). ROS
1991; Jain et al., 1998). (Figure 2) can react with a spectrum of metabolites,
Both in initiating cultures and in sub-culturing, proteins including enzymes, and nucleic acid mo-
explant preparation involves wounding of the tissues lecules (Gille and Siegler, 1995). Oxidised enzymes
which is known to cause oxidative stress (Yahraus et which may be inactivated, are degraded by cytoso-
al., 1995). Elicitors of oxidative stress e.g. hypochlor- lic proteinases (Laval, 1996). The influence of ROS,
ite (Wiseman and Halliwell, 1996) and mercuric salts through altered cell redox potential, on the cell cycle
(Patra et al., 1997), are used to surface sterilize the and oxidative damage to both nuclear and organellar
 147

Figure 2. Reactive oxygen species (ROS) produced constitutively in the cell. The upper section shows the natural antioxidants and enzymes
used to minimize the toxic effects of ROS. The lower section gives selected examples of the harmful effects of ROS when the pro- and
anti-oxidant balance is perturbed in oxidative stress. (MDE, malondialdehyde; HNE, 4-hydroxynonenal).

DNA, may result in mutations (Figure 3; Bohr and 1995). Such changes could explain, at least in part, the
Dianov, 1999). Oxidative damage in eukaryote cells range of variability found in plant cells, tissues and
is expressed in altered hyper- and hypomethylation of organs in culture and in microplants, namely, recal-
DNA (Kaeppler and Phillips, 1993; Tilghman, 1993; citrance including loss of cell competence (Hagege,
Wiseman and Halliwell, 1996; Cerda and Weitzman, 1995; Lambe et al., 1997), hyperhydricity (Olmos et
1997; Wacksman, 1997); changes in chromosome al., 1997) and somaclonal variation including epigen-
number from polyploidy to aneuploidy, chromosome etic and genetic variation (Jain et al., 1998; Joyce et
strand breakage, chromosome rearrangements, and al., 1999; Kowalski and Cassells, 1999).
DNA base deletions and substitutions (Gille et al., The objective of this review is to discuss tissue cul-
1994; Czene and Harms-Ringdahl, 1995; Hagege, ture variability, its causes, detection and remediation
148

with emphasis on the possible role of oxidative stress

in this phenomenon.

Aberrations and variation expressed in vitro

At the outset it should be recognised that explants,

other than buds, from dicot plants have different
characteristics to those from monocots, specifically
those of dicots may have a cambium; that there are
differences in organogenetic potential between famil-
ies, genera, species and genotypes; and that different
genotypes of a species may show widely different re-
sponses (George, 1993, 1996). Further there are differ-
ences in the responses of explants from different parts
of a plant, which change ontogenetically (George, loc. Figure 3. Changes in DNA caused by oxidative stress which
cit.). Some trends are evident, e.g. increased recal- can lead to recalcitrance, loss of competence, hyperhydricity and
somaclonal variation.
citrance with advancing age of the cultures (Hagege,
1995) and increased somaclonal variability in micro-
plants with increasing sub-culture number (Brar and
Jain, 1998). With a given genotype, wounding of the selection for in vitro conditions may result in loss of
tissues on cutting (excision), and tissue damage and competence; e.g. selection based on fitness of grossly
exposure to sterilants during sterilisation, and sub- altered genotype(s) may result in the irreversible loss
optimal in vitro factors (Ziv, 1991) are important in of competence (Hagege, 1995).
relation to genomic damage. So called ‘pre-existing’ The main morphological aberration seen in shoots
genomic diversity at the cell level (D’Amato, 1964; in vitro cultures, both in nodal/bud derived shoots
Figure 4) and wound or oxidative damage due to and in adventitious shoots, is hyperhydricity (‘vitri-
wounding may explain some of the variability sub- fication’) (Debergh et al., 1992). This term is used
sequently seen in vitro and in the resulting micro- to describe aberrant morphology, typically hyperhy-
plants. Possible stress due to unbalanced media, bad drated, translucent tissues and physiological dysfunc-
culture vessel design and environmental stress may tion in plant tissues in vitro (Ziv, 1991). It is also
also, or further, contribute to the genetic, epigenetic associated with leaf-tip and bud necrosis. The latter
(developmental) and physiological variability recor- often leads to loss of apical dominance in the shoots
ded (Ziv, 1991). Wounding or excision per se may be and is associated with callusing of the stem base.
considered both as a trigger for cell division (Sangwan An important characteristic of this condition is im-
et al., 1992) and as a damaging oxidative burst (Schaaf paired stomatal function which causes problems in
et al., 1995; Yahraus et al., 1995). establishing microplants (Preece and Sutter, 1991).
As a consequence of the above factors, explants Morphological variability in plants from in vitro
may senesce, fail to respond, undergo cell division culture may be seen in intrapopulation variability
and/or produce adventitious organs or somatic em- (within a population of adventitiously regenerated
bryos. In responding genotypes, the response is gen- plants) (Kowalski and Cassells, 1998) and interpop-
erally regulated in a predictable way by manipulation ulation variability (between populations of in vitro
of the auxin to cytokinin ratio and absolute growth plants) (Joyce et al., 1999). The latter may arise when
regulator concentrations (Skoog and Miller, 1957). In plants are propagated on different media or in cul-
some cases, recalcitrance may be overcome by pulsing ture vessels with different characteristics (Joyce et al.,
in sequence with auxin followed by cytokinin (Chris- 1999). Intrapopulation variability can be a result of the
tianson and Warnick, 1985). Whether genome vari- loss of specific viruses, including cryptic viruses, from
ability is ‘pre-existing’, caused by oxidative stress on some of the regenerated plants (Matthews, 1991); chi-
wounding an/or caused by stress in culture (Figure 4), meral breakdown, rearrangement and/or synthesis of
selection may begin in vitro with the appearance of unstable chimeral plants (Tilney-Bassett, 1986). In
sectoring in the callus (D’Amato et al., 1980). Cell line generally, heritable somaclonal variation (Larkin and
 149

Figure 4. Sources of genetic variation in plants obtained through organogenesis in callus cultures (reproduced with permission; from George,
1993).

Scowcroft, 1981) has the characteristics of mutation etic/ontogenetic variability) may be more susceptible
(Anonymous, 1995; Jain et al., 1998), albeit occur- to damping-off diseases (Agrios, 1997). This is not
ring at higher frequency than occurs spontaneously always the case, as juvenile tissues are reported to
in seed or vegetative propagules (Preil, 1986). It is have enhanced resistance to fusaric acid (Barna et al.,
genotype-dependent and dependent on the pathway of 1995) and Cassells et al. (1991) have shown that potato
regeneration (Karp, 1991). crops derived from microplants, showing juvenility
Epigenetic changes can occur in vitro culture res- compared to a tuber-derived crop, were more resist-
ulting in ‘apparent rejuvenation’ (Pierik, 1990) af- ant to potato blight. In vitro plants may have a longer
fecting woody and herbaceous plants (Huxley and time to flowering compared to those from vegetative
Cartwright, 1994; James and Mantell, 1994; Jemmali propagules (Cassells et al., 1999a). While morpholo-
et al., 1994; Cassells et al., 1999 a, b). Interpopula- gical intrapopulation variability and ontogenetic and
tion variation is usually cryptic, as control populations physiological variation, expressed in interpopulation
are not available for comparison; it is recognised in variability, are well recognised phenomena in mi-
quality differences in plants produced by different pro- cropropagation, cryptic intrapopulation variation in
tocols or by different micropropagators (Grunewaldt- juvenility has also been detected in adventitiously re-
Stoker, 1997). Examples of interpopulation variability generated plant populations showing genetic variation
are populations differing in degree of hyperhydri- (Kowalski and Cassells, 1998) suggesting that genetic
city or juvenility (Swartz, 1991). While woody plant and epigenetic variability are not necessarily discrete
propagators are familiar with phase change (How- but can occur in the same population.
ell, 1998), micropropagators of herbaceous plants Somaclonal variation is strongly expressed after
appear less conscious of this phenomenon but it has the microplant population establishment stage as inter-
implications for disease susceptibility in that poly- plant variation in morphological characters. Some of
genic resistance develops as the plant soma matures the plants may show characteristics of chimeral break-
(Agrios, 1997) and for time to flowering (Howell, down (Tilney-Bassett, 1986). Somaclonal variation
1998). Plants showing prolonged juvenility (epigen- has been extensively reviewed in Jain et al. (1998).
150

Figure 5. Showing the consequences of oxidative stress from in-

duction of host antioxidant defences (repair, heat shock protein
induction, pathogenesis related protein induction) to mutation,
programmed cell death and uncontrolled cell death.

Figure 6. The relationship between stress inducers e.g. medium

As discussed above, shifts in characters in popula- salt stress, gene activation and the generation of biomarkers for
stress remediation and stress damage repair. Examples of damage
tions, e.g. physiological or developmental changes, are exposure are ethylene and ethane; of damage are oxidised bases
not readily recognised unless control populations are e.g. 8-oxoguanine; of remediation are glutathione and glutathione
available (Cassells et al., 1997). These can, however, reductase and of repair, Poly(ADP-ribose) polymerase (see text for
be visually expressed in loss of apical dominance, leaf further markers).
number and leaf size and, more importantly in the time
to flowering, and yield quality e.g. tuber number and Klerk, 1990) and can be quantified using computerised
size distribution in potato seed production (Cassells et image analysis (Cassells et al., 1999a).
al., 1999a). Analysis of DNA-base methylation and various ge-
netic fingerprinting techniques have also been used to
confirm and characterise variability in tissue culture-
Characterisation of epigenetic and genetic changes derived plants, confirming both morphological and
in microplants pre- and post- establishment cryptic genetic and epigenetic variability between and
within populations (Karp et al., 1998; Cassells et al.,
Cytometric analysis of callus has shown variability 1999b).
in chromosome number and ploidy in tissue culture-
derived plants (Geier, 1991; Gupta, 1998). Investiga-
tions indicate more chromosome variability in the cal- Current views on the molecular basis of
lus phase than in adventitious shoots (D’Amato et al., somaclonal variation
1980), indicating a loss of competence in the more ser-
iously disturbed genomes (Valente et al., 1998). Cell In recent years plant cell, tissue and organ culture has
line selection for secondary product formation also been developed for applications in plant genetic ma-
shows differences at the metabolite level (Berglund nipulation (Cassells and Jones, 1995). In this field,
and Ohlsson, 1995). somaclonal variation has attracted considerable in-
While occasional albino shoots are observed, the terest as a means of improving crop plants (Jain et
expression of morphological variation is difficult to al., 1998). Reviews discussed a number of mechan-
assess in vitro due to variability between shoots due to isms to explain somaclonal variation, these included
temporal differences in shoot initiation and because of changes in chromosome number, chromosome break-
the limited leaf expansion in in vitro cultures. Variab- age and rearrangement, DNA amplification, point
ility in both qualitative and quantitative traits has also mutations, changes in DNA methylation, changes in
been reported (Karp, 1991). The latter expressed in in- organellar DNA, activation of transposons (Frahm et
creased standard deviations of the character mean (De al., 1998; Gupta, 1998; Henry, 1998; Jain et al.,
 151

1998; Kaeppler et al., 1998). The mechanisms ap- (Figure 5). In addition to the above ROS, chemical
pear to be equally applicable to explaining the basis and physical agents can stimulate lipid peroxidation
of variation at the cell and callus level and are sim- that can become autocalatytic resulting in the produc-
ilar to the variability resulting from oxidative genome tion of organic hydroperoxides (Figure 2). ROS in
damage and induced mutation. Nagl (1990) has dis- the presence of iron and copper ions may generate
cussed the relationship between stress-induced and highly mutagenic compounds, e.g. peroxyl radicals
ontogenetic changes in plant genomes arguing that and alkoxyl radicals (Koh et al., 1997).
plant genomes are inherently fluid. In some genomes, The primary response to elevated ROS production
e.g. flax (Schneeberger and Cullis, 1991) and banana (Figure 2) is stimulation of production of antioxid-
(Cullis and Kunert, 2000) there are well character- ant molecules (radical scavengers) such as ascorbic
ised genomic instabilities associated with somaclonal acid and glutathione in the aqueous phase and alpa-
variation. tocopherol and carotenoids in the lipid phase (Gille
and Sigler, 1995) and the activation of antioxid-
ant enzyme systems including superoxide dismutase,
The relationship between somaclonal variation catalase and glutathione and ascorbic acid peroxidases
and spontaneous mutation (Tsang et al., 1991; Larson, 1995; Smirnoff, 1996).
Additional responses involve the activation of heat
The paper of Shepard et al. (1980) on somaclonal vari- shock proteins to protect enzymes systems against
ation in potato stimulated interest in the application ROS damage (Burdon, 1993) and of proteases to
of this variability in crop improvement but was soon degrade damaged proteins (Stadtman, 1992). More
followed by concern about the quality of somaclonal significant is the potential of ROS to cause DNA dam-
variation and whether it differed qualitatively from age (‘genotoxicity’; Wiseman and Halliwell, 1996).
spontaneous mutation (Sanford et al., 1984). This is- The cell cycle slows or shuts down to minimise the
sue has been discussed by Karp (1991, 1995) but while transmission of mutations to daughter cells through
it is still controversial, somaclonal variation is used in mitosis and to facilitate DNA repair (Logemann et
plant improvement, with induced mutagenesis whose al., 1995; Amor et al., 1998; Reichheld et al., 1999)
efficiency has been improved by exploiting in vitro and DNA repair mechanisms, the SOS response, are
plant systems (Cassells, 1998). More importantly here, activated (Laval, 1996; Yamamoto et al., 1997; Von-
induced mutation and somaclonal variation result in arx et al., 1998). The outcomes of oxidative stress
a qualitatively similar, if not quantitatively identical, depend on the balance between pro and anti-oxidants
spectrum of DNA changes (see Figure 3). The issue is responses. Imbalance may lead to controlled responses
whether somaclonal variation and other tissue culture (Figure 5) such as induced resistance to pathogens
variability are mechanistically like physically-induced (Ernst et al., 1992), excessive imbalance to cell dam-
mutation and are caused by reactive oxygen species age and mutation (Wiseman and Halliwell, 1996),
(Anonymous, 1977; 1995; Micke and Donini, 1993). possible programmed cell death (apoptosis) (Polyak
et al., 1997) and, in the extreme, to (unprogrammed)
cell death (Hippeli and Elstner, 1996). Oxidative stress
Oxidative stress and mutation has been linked to recalcitrance in protoplast culture
(Benson and Roubelakis-Angelakis, 1994).
Oxidative stress is caused by the unremediated hyper- Increase in ROS is associated with a range of
activity of reactive oxygen species (Figure 2). ROS biotic and abiotic stresses (Gile and Siegler, 1995;
such as superoxide, hydrogen peroxide and the hy- Bartosz, 1997). These include salt, drought, heat,
droxyl radical are metabolic intermediates in respira- and UV-induced stresses. They are also induced by
tion and photosynthesis and other metabolic activities chemical and physical mutagens (Anon, 1977). Their
in plants (see review by Gille and Sigler, 1995; Bar- protective and constitutive roles include direct protec-
tosz, 1997). Their natural cytoplasmic toxicity and tion against pathogen attack, and involves the role of
genotoxicity is controlled by antioxidants and en- H2 O2 as a messenger in the induction of host res-
zymic pathways in the cell (Hippeli and Elstner, 1996). istance (pathogenesis-related (PR) protein induction)
Various environmental signals (Bartosz, 1997), which (Lamb and Dixon, 1997). H2 O2 may have a role
lead to an increase in ROS are associated with in- in xylem formation and other cell-death processes in
duced mechanisms to minimise their harmful effects plants (Howell, 1998). ROS have a role in creating
152

variability in the plant genome by activating transpo- number and DNA content (Quicke, 1993; Curry and
sons (Mhiri et al., 1997), inducing polyploidy, chro- Cassells, 1998). Techniques including fluorescent in
mosome breakage/rearrangements and base mutations situ hybridisation (FISH) (Maluszynska and Heslop-
(Figure 2). DNA based changes induced by ROS may Harrison, 1991) and Giemsa banding (Quicke, 1993)
inhibit methylating enzymes leading to hypomethyla- are used to look for somatic recombination, includ-
tion (Cerda and Weitzman, 1997; Wacksman, 1997), ing chromosome breakage and rearrangement and may
e.g. formation of 8-oxoguanine occurs at high fre- also be used to detect DNA amplification and re-
quency which may lead to mismatch at DNA repair duction. Changes in DNA base methylation can be
(base mutation, e.g. AT–GC changes), similarly for investigated using methylation-sensitive restrictions in
other oxidative base changes. RFLP and AFLP analysis (Karp et al., 1998) or by
While much of the ROS-induced effects due to PCR of bisulphite modified DNA (Joyce et al., 1999).
wounding may be localised, some ROS can migrate
across membranes, e.g. H2 O2 and cause effects dir-
ectly, or via membrane lipid peroxidation, at the Plant hormones and oxidative stress
level of DNA in the stressed cells or in neighbouring
cells (Figure 2). A gradient of stress damage from Plant hormones implicated in hyperhydricity (vit-
the wound area back into the explant may be hy- rification) include cytokinins, auxins, and the
pothesised. ROS (and physical mutagens) have been auxin/cytokinin ratio; gibberellic acid and ethylene
confirmed in animal cells (Halliwell, 1999) as causing (Ziv, 1991; George, 1996). Oxidative stress has been
the range of epigenetic and genetic changes in DNA associated with auxin and cytokinin metabolism in
that are problematic in plant tissue culture (Figure 3). Agrobacterium induced tumours (Jia et al., 1996).
Ethylene is also strongly linked to oxidative stress
(McKersie and Leshem, 1994; Pell et al., 1997). In-
Parameters used to characterise oxidative stress jury has been shown to activate the oxidation of IAA,
while kinetin is reported to be a secondary product of
A number of methods have been used to mon- oxidative stress (Barciszewski et al., 1997).
itor/characterise oxidative stress (Figure 6). These in- In vitro, various media factors have been shown to
clude measurement of the redox potential (Reichheld induce stress, including hormones, and mineral nutri-
et al., 1999), measurement of stress related metabol- ents (Ziv, 1991; George, 1993, 1996) there is evidence
ites e.g. ascorbic acid (Smirnoff, 1996), glutathione that metal toxicities and deficiencies may generate
(de Vos et al., 1994), hydrogen peroxide (Schreck et ethylene through oxidative stress (Lynch and Brown,
al., 1996). Ethylene has been monitored in hyperhy- 1997). Oxidative stress also affects cytosolic calcium
dricity (‘vitrification’) studies (Keevers and Gaspar, (Price et al., 1994). Oxidative stress has been sugges-
1985) and along with ethane, a marker for lipid per- ted as a cause of guard cell malfunction (McAnish et
oxidation, has been used to monitor stress in vitro al., 1996). Calcium has been implicated in increased
(Cassells et al., 1980; Cassells and Tamma, 1985). stress tolerance (Gong et al., 1997).
Thiobarbituric acid reactive substances are also used
to assess lipid peroxidation (Laszczyca et al., 1995). 8-
oxo-2-deoxyguanosine (Kasai, 1997; Bialkowski and Remediation of oxidative and other tissue culture
Olinski, 1999) is considered to be a reliable indicator associated stresses
of genotoxicity as are other bases modified by ROS
(Yamamoto et al., 1997). Enzymes of oxidative meta- Remediation of oxidative stress can be based on sev-
bolism (Mehlhorn, 1990), enzymes associated with eral strategies. Genotypes can be screened for their
the cell cycle (Chiatante et al., 1997), enzymes of the sensitivity to stress and sensitive genotypes avoided;
SOS response (Laval, 1996; Wiseman and Halliwell, or they can be bred, mutated or engineered for in-
1996) e.g. poly(ADP-ribose)-polymerase (Amor et al., creased stress tolerance (Gupta et al., 1993). The
1998), screening for heat-shock proteins (HSP) (Bur- breeding/genetic manipulation options are both relat-
den, 1993) and PR proteins (Glandorf et al., 1997) ively long-term and costly and can only be applied to
have also been used as oxidative stress monitors. individual genotypes (Jones and Cassells, 1995).
Karyotyping, flow cytometry and microdensito- An important consideration where plant tissue cul-
metry can be used to measure changes in chromosome ture is used for cloning or transformation is the choice
 153

of the explant. While mature plant tissue may be poly- Manipulation of media composition, particularly
somatic (D’Amato, 1964), this may not be so in the using simple media formulations in autotrophic or
case of the tissues of young plants in vitro (Curry microhydroponic culture (Cassells, 2000a) and modi-
and Cassells, 1998; Curry and Cassells, unpublished). fication of culture vessel design to facilitate controlled
Selection of explants from the latter may avoid the gaseous exchange (Cassells and Walsh, 1998), can
problem of ‘pre-existing’ variation (Figure 4). The greatly influence microplant resistance to hyperhydri-
main option, however, is the use of stable pathways city and improve ontogenic development (Cassells,
of multiplication (Figure 1: George, 1993, 1996); al- 2000b).
beit, even with these genetic drift may occur (Jemmali
et al., 1997). There is evidence e.g. that protoplasts
give rise to greater variability than tissue explants as
expressed in the greater variability in plants regen- Conclusions
erated from the former (Sree Ramulu et al., 1984).
This may reflect expression of somatic cell variabil- It is speculated here that problems underlying the ap-
ity (Figure 4) but it also appears to reflect the stress plication of plant tissue culture systems in plant clon-
experienced in their isolation and regeneration where ing (micropropagation) and genetic transformation,
they are exposed to osmotic stress and the toxic effects namely, recalcitrance, hyperhydricity, poor physiolo-
of cell wall degrading enzyme preparations (Cassells gical quality, genetic and epigenetic variation, may
and Tamma, 1985) and this has implication for their have a common basis, at least in part, in oxidat-
use in transformation via electroporation or biolistics ive stress-induced damage. This damage is caused
at the protoplast level (Christou, 1995) as opposed to by an overwhelming of the antioxidative defenses by
bombardment of apices (Sautter et al., 1995). primary ROS and secondarily, by the production of
The immediacy of the oxidative stress caused ROS by lipid autocatalytic peroxidation (Figure 2).
by excision and manipulation (Yahraus et al., 1995) While damaged proteins may be broken down by
would suggest that treatment of the explant after ex- proteinases, damage is fixed in the DNA. Specific
cision would be relatively ineffectual. As an alternat- levels of DNA damage may result in cell death or
ive, it is suggested that stress be applied to the donor programmed cell death, other levels in loss of cell
plant (or in vitro microplant) before excision. Under in competence, altered methylation patterns may result
vivo conditions, it was been shown that stress exposure in epigenetic changes, or in mutations (Figure 3).
induces cross-stress tolerance, e.g. UV treatment not Somaclonal variation shows a similar spectrum of ge-
only increases tolerance to further UV exposure but netic variation to induced mutation; and oxidative
also to pathogen induced stress (Ernst et al., 1992), stress and irradiation are known to involve ROS.
similarly paclobutrazol which is used in vitro and Oxidative stress damage which it is emphasised is
in weaning treatments (George, 1993, 1996) reduces genotype dependent, may also operate at the physiolo-
oxidative stress to high light and high temperature gical level since ROS have been shown to influence
(Mahoney et al., 1998). It has been suggested that plant hormones namely, cytokinin, auxin, ethylene
induction of heat shock proteins be used to protect metabolism. Oxidative stress also influences calcium
against oxidative stress (Banzet et al., 1998; Sebehat metabolism which in turn is involved in auxin trans-
et al., 1998). port, guard cell function and as a secondary messen-
Prevention or reduction of oxidative stress dam- ger.
age in vitro may be possible by manipulating hormone Remediation strategies have been proposed and
and mineral nutrients using the above oxidative stress some makers of oxidative stress listed. It is sugges-
parameters to monitor the protocols as has been done ted that induction of heat shock proteins may confer
in the case of protoplasts (Cassells et al., 1980; Cas- cross tolerance to the stress phenomena encountered
sells and Tamma, 1985). In vitro calli and tissues as problems in establishing plant tissue cultures. Me-
are more amenable than intact tissues to permeation dia and environmental manipulations should be carried
techniques. Cells, calli and explants can be infiltrated out aimed at reducing in vitro stress based on adjusting
and bathed with radical scavengers such as ascorbic the media composition and the physical culture envir-
acid, mannitol and dimethyl sulphoxide. The stress onment, paying special attention to hormone stress,
messenger hydrogen peroxide can be broken down by mineral composition and water and light stress. Re-
extracellular catalase. active nitrogen species (NOS) not discussed here, can
154

parallel the stresses caused by ROS (Wiseman and Bialkowski K & Olinski R (1999) Oxidative damage to plant DNA
Halliwell, (1996). in relation to growth conditions. Acta Biochim. Pol. 46: 43–49
Bohr VA & Dianov GL (1999) Oxidative DNA damage processing
Recalcitrance, hyperhydricity, somaclonal vari- in nuclear and organellar DNA. Biochimie 81: 155–160
ation and oxidative stress in tissue culture are com- Bolwell GP, Butt VS, Davies DR & Zimmerlin A (1995) The origin
plex, multifactorial phenomena based on genotype, of the oxidative burst in plants. Free Rad. Res. 23: 517–532
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